Mycobacterium tuberculosis is a tough bug to kill. The sterilization of a M. tuberculosis infection in a patient with active tuberculosis requires a minimum of 6 months of treatment with 3-4 drugs taken daily. This tenacity is unparalleled in any other human bacterial pathogen. M. tuberculosis has evolved strategies to survive in the face of drugs and the host immune responses. The current dogma in the field suggests that survival is the result of the bacteria's ability to enter into a drug tolerant state, where it is non-replicative and dormant, but clearly viable. If drugs are stopped early or the immune system is compromised, dormant bacteria can revive and go on to active disease. As part of the program project, we are dedicating our time and resources to find the "Achilles'heel" of the dormant bacteria using mycobacterial genetics. For this proposal, we have generated a high throughput methodology of specialized transduction, which can be used to disrupt any nonessential gene. In addition, we have used conditional expression and specialized transduction to develop CESTET, a method to prove essentiality and to study a mycobacterial cell's fate upon depletion of the gene product. In vitro and in vivo assays have been developed to screen for essential genes and persistence factors. The group has a proven track record of successfully solving structures having solved 70% of the Mtb structures in the PDB, and we will build on this success to solve the structures of the new essential or persistence targets. Using the structural information, virtual ligand screens will be performed to identify new inhibitors. Thus, the goal of this Project is to validate targets, solve their three dimensional structures, and propose novel inhibitors for further drug development to radically shorten the time to treat TB.